Abstract

Previous studies have shown that repression of the Saccharomyces cerevisiae SER3 gene is dependent on transcription of SRG1 from noncoding DNA initiating within the intergenic region 5' of SER3 and extending across the SER3 promoter region. By a mechanism dependent on the activities of the Swi/Snf chromatin remodeling factor, the HMG-like factor Spt2, and the Spt6 and Spt16 histone chaperones, SRG1 transcription deposits nucleosomes over the SER3 promoter to prevent transcription factors from binding and activating SER3. In this study, we uncover a role for the Paf1 transcription elongation complex in SER3 repression. We find that SER3 repression is primarily dependent on the Paf1 and Ctr9 subunits of this complex, with minor contributions by the Rtf1, Cdc73, and Leo1 subunits. We show that the Paf1 complex localizes to the SRG1 transcribed region under conditions that repress SER3, consistent with it having a direct role in mediating SRG1 transcription-dependent SER3 repression. Importantly, we show that the defect in SER3 repression in strains lacking Paf1 subunits is not a result of reduced SRG1 transcription or reduced levels of known Paf1 complex-dependent histone modifications. Rather, we find that strains lacking subunits of the Paf1 complex exhibit reduced nucleosome occupancy and reduced recruitment of Spt16 and, to a lesser extent, Spt6 at the SER3 promoter. Taken together, our results suggest that Paf1 and Ctr9 repress SER3 by maintaining SRG1 transcription-dependent nucleosome occupancy.

The Paf1 complex colocalizes with actively transcribed SRG1. (A) ChIP analysis of HA-tagged Paf1 complex subunits at SRG1 (SER3-22 and SER3-19) and the flanking AIM9 (SER3-41) and SER3 (SER3-1) genes from untagged (FY4), 3×HA-PAF1 (KY1721), 3×HA-RTF1 (KY2082), LEO1-3×HA (KY785), and 3×HA-CDC73 (KY786) strains grown in YPD at 30°C. (B) ChIP analysis of Ctr9-Myc from untagged (KY399) and CTR9-6×MYC (KY785) strains grown in YPD at 30°C. The relative occupancies of these factors were calculated using qPCR as described in Materials and Methods. Each value represents the mean ± the standard error of the mean of three biological replicates, and asterisks indicate statistical significance compared to the untagged control (*, P < 0.05; **, P < 0.01). Below the graphs is a schematic of the SRG1/SER3 locus, and the arrows indicate the transcription start sites of SRG1 and SER3. The gray box represents the Cha4 binding site, black boxes indicate TATA sequences, and white boxes are sequences required for SER3 activation. The block arrow indicates SRG1 transcription, and the horizontal black bars mark the location of the DNA fragments amplified by qPCR.

Paf1 and Ctr9 are required for nucleosome occupancy over the SER3 promoter. (A) Nucleosome scanning assays were performed on wild-type (FY4), leo1Δ (KY1805), cdc73Δ (KY1706), rtf1Δ (KY1703), ctr9Δ (KY2170), and paf1Δ (KY1700) strains grown in YPD at 30°C. MNase protection across the SER3 locus was calculated relative to a positioned nucleosome within the GAL1 promoter by using qPCR as described in Materials and Methods. The mean ± standard error of the mean from three biological replicates is plotted at the midpoint for each PCR product. Shown below the graph is a diagram of the SER3 locus, comparing the positions of nucleosomes (gray ovals) extrapolated from the MNase protection data between wild-type and ctr9Δ or paf1Δ strains. The light gray ovals are indicative of reduced nucleosome occupancy compared to the darker ovals (wild-type strains). (B) ChIP analysis of histone H3 from wild-type (FY4), paf1Δ (YJ1030), ctr9Δ (YJ1016), and rtf1Δ (YJ1014) strains grown in YPD at 30°C. The relative occupancies of these factors were calculated as described in Materials and Methods. Each value represents the mean ± the standard error of the mean of at least three biological replicates, and asterisks indicate statistical significance compared to wild type (*, P < 0.05; **, P < 0.01). The P values for the decrease in H3 occupancy over primer set SER3-22 in paf1Δ and ctr9Δ strains were 0.07 and 0.06, respectively. Below the graphs is a schematic of the SRG1/SER3 locus, with the arrow indicating SRG1 transcription and the black bars indicating the location of the DNA fragments amplified by qPCR.

Paf1 and Ctr9 are required for Spt6 and Spt16 colocalization to actively transcribed SRG1. ChIP of Spt6-FLAG (A), Spt16 (B), and Rpb3-HA (C) from untagged (FY4) strains and wild-type (YJ882), paf1Δ (YJ1031), and rtf1Δ (YJ1013) strains that all express epitope-tagged versions of Spt6 (SPT6-FLAG) and Rpb3 (RPB3-3×HA). The relative occupancies of these factors were calculated using qPCR as described in Materials and Methods. Each value represents the mean ± the standard error of the mean of three biological replicates, and asterisks indicate statistical significance compared to the wild type (*, P < 0.05; **, P < 0.01). Below the graphs is a schematic of the SRG1/SER3 locus, with the arrow indicating SRG1 transcription and the black bars indicating the location of the DNA fragments amplified by qPCR. (D) Western analysis of Spt6 and Spt16. The wild-type, paf1Δ, ctr9Δ, and rtf1Δ strains shown in panel A were subjected to Western blotting to compare Spt6 (top panel) and Spt16 (bottom panel) protein levels between these strains. Representative immunoblots are shown. These blots were reprobed with an antibody to G6PDH as a loading control. (E) Quantitation of Western analyses from a minimum of four biological replicates. The values shown are the mean Spt6-FLAG (black) and Spt16 (gray) protein levels, normalized to the G6PDH loading control and made relative to the wild-type strains. Error bars indicate standard errors of the means, and asterisks indicate statistical significance compared to the wild type (*, P < 0.05; **, P < 0.01).

Effects of the Paf1 complex mutant on histone H3, Spt6, and Spt16 occupancy at other transcribed genes. (A and B) Relative occupancies of HA-Paf1, HA-Rtf1, Leo1-HA, and HA-Cdc73 (A) and of Ctr9-MYC (B) within the coding sequence of two highly transcribed genes, PMA1 and ADH1, and two lowly transcribed genes, GAL1 and CYC1, were determined by qPCR using the ChIP assay shown in Fig. 3. (C to E) Relative occupancies of Spt6-FLAG (C), Spt16 (D), and Rpb3-HA (E) at these four genes were determined by qPCR by using the immunoprecipitated chromatin shown in Fig. 6. (F) Relative occupancy of histone H3 was determined by qPCR using the immunoprecipitated chromatin assayed in Fig. 5B. The occupancies of each of these factors at these genes were normalized to their occupancies at a control region on chromosome V that contained no open reading frames (no ORF). Each value represents the mean ± standard error of the mean for at least three biological replicates, and asterisks indicate statistical significance (*, P < 0.05; **, P < 0.01). (G and H) Spt6-FLAG (G) and Spt16 (H) ChIP data from wild-type, paf1Δ, and rtf1Δ strains (C and D) were normalized to Rpb3-HA ChIP data (E).